CN104327139B - Preparation method of cordycepin crystal - Google Patents
Preparation method of cordycepin crystal Download PDFInfo
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- CN104327139B CN104327139B CN201410507918.5A CN201410507918A CN104327139B CN 104327139 B CN104327139 B CN 104327139B CN 201410507918 A CN201410507918 A CN 201410507918A CN 104327139 B CN104327139 B CN 104327139B
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- cordycepin
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- crystal
- cordyceps militaris
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Abstract
The invention discloses a preparation method of a cordycepin crystal. The preparation method includes following steps: (1) with residual substrate after cultivation of cordyceps militaris stroma as a raw material, crushing the substrate, performing extraction with an organic solvent and performing centrifugation to collect a supernate; (2) performing purification to a crude liquid after the centrifugation through a macroporous resin adsorption chromatographic column; (3) performing enrichment to the cordycepin obtained through the step (2) with an aqueous two-phase system composed of ethanol and ammonium sulfate; and (4) performing elution in a chromatographic column with a methanol solution, performing reduced pressure distillation for concentrating an eluent, performing methanol elution to the concentrated eluent, and performing crystallization to the eluent to obtain the cordycepin crystal. The preparation method of the cordycepin crystal is reduced in production cost, increases the purity of the cordycepin and is free of residual organic solvent.
Description
Technical field
The present invention relates to the preparation method of cordycepin and its crystal, belong to medicinal chemistry arts.
Background technology
Cordyceps militaris is Ascomycotina, ergot Zoopagales, Clavicipitaceae, the type sepecies of Cordyceps, and scientific name is
Cordycepsmilitaris, also known as northern Chinese caterpillar Fungus, Cordceps militaris etc., the traditional Chinese medical science thinks, Chinese caterpillar fungus enters lung kidney two warp, can tonifying lung
The moon, and energy kidney-replenishing, cure mainly kidney deficiency, impotence and seminal emission, soreness of waist and knee joint, eak after being ill, chronic cough is weak, phthisical cough phlegm blood, spontaneous sweating
Deng being a kind of unique Chinese medicine that can balance, adjust negative and positive, be widely used simultaneously.
Cordycepin is the major physiological active component of Cordyceps militaris, is a kind of nucleosides material, by adenine and deoxypentose
Composition, it has very big market prospects in terms of health care, and the extraction of current cordycepin is all to pass through again from Cordyceps militaris
Miscellaneous technique is extracted, and has water-bath, and alcohol bathes extraction, resin adsorption method, chromatography method, active carbon adsorption etc., these sides
Or method complex operation, recovery rate are low, or through different resin treatment or color need to must be prepared by expensive efficient liquid phase
Spectral technology equipment, production cost height is it is difficult to realize scale industrial production.
This area once attempted obtaining highly purified plate-like cordycepin crystal with the residue cultivating Cordyceps militaris in recent years, but this
Plant method yield relatively low, there is larger wastage of material.
Content of the invention
For defect of the prior art, the invention discloses a kind of preparation method of cordycepin crystal, to cultivate pupa worm
Remaining matrix after grass seed seat be raw material it is achieved that the recycling of waste, and simple to operate, mild condition, recovery rate is high, easily
In expanding production, there is not organic problem of solvent residual, and the cordycepin crystal purity being obtained is high.
For achieving the above object, the present invention is achieved through the following technical solutions：
The preparation method of cordycepin, comprises the steps：1) to cultivate the remaining matrix of Cordyceps militaris stroma as raw material, by base
Matter is extracted with organic solvent after pulverizing, and supernatant is collected by centrifugation；2) utilize macroporous resin adsorption chromatographic column to centrifugation after
Thick liquid is purified；3) step 2) gained cordycepin using ethanol, ammonium sulfate composition double-aqueous phase system be enriched with and collected
Sample；4) fill chromatographic column, methanol solution elutes, and eluent vacuum distillation concentrates, the eluant methanol wash-out of concentration, eluent is tied
Crystalline substance obtains cordycepin crystal.
In above-mentioned preparation method, remaining matrix is cultivated with Cordyceps militaris stroma and reduces entirely for raw material extraction cordycepin
The production cost of process, and achieve the twice laid of remaining matrix, improve the added value of product of Cordyceps militaris；Using organic solvent
To cordycepin, thick immersion proposes relatively conventional water-bath extraction, and in leaching liquor, cordycepin content is higher, and impurity is less；Using macropore tree
Fat adsorption chromatography post purifies to sample, can realize at normal temperatures continuously dynamically separating, simple to operate, low cost, receives
The cordycepin purity liquid of collection is high；Double-aqueous phase system using ethanol, ammonium sulfate composition is enriched with to cordycepin, and extraction is completeer
Entirely, product purity is higher.
For generally, the method for the present invention adopts macroreticular resin absorbing method to combine ethanol, ammonium sulfate aqueous two-phase extraction skill
Art, compared with traditional flooding, macroreticular resin absorbing method, simple to operate, product purity is higher, be suitable to expanding production and one
There is not organic problem of solvent residual, environmental protection.
Wherein, the described culture remaining matrix of Cordyceps militaris stroma refers to exist for the solid medium cultivating Cordyceps militaris stroma
Remove the remaining matrix of Cordyceps militaris stroma after the completion of culture, such matrix main component one be wheat or other pupa worm can be provided
The composition of grass seed seat growth.
In the present invention, described matrix is pulverized standard and is crossed 80 mesh sieves for after pulverizing, and organic solvent used is ethanol solution, with
The envelope-bulk to weight ratio of matrix is 2-10: 1, and extraction temperature is 50-80 DEG C, and extraction time is 3-8 hour.
Generally, above-mentioned ethanol solution concentration is the ethanol water of 40-75%, the degree that above-mentioned matrix is pulverized with used
Organic solvent match, maximized can improve extracting efficiency.
Wherein, chromatography parameter be cordycepin sample liquid and macroreticular resin with 50ml: 0.8-1.5g amount loading,
1.5-2.0BV/h, then uses the distilled water removal of impurities of 3-8BV, and flow velocity is 1.5-2.0BV/h, is finally washed with the ethanol solution of 3-8BV
De-, flow velocity is 1.5-2.0BV/h.
Wherein, ethanol solution concentration used is the ethanol water of 40-75%.
Preferably, chromatography parameter is cordycepin sample liquid and macroreticular resin with 50ml: 1g amount loading, (now
PH is about 5), 1.8BV/h, then uses the distilled water removal of impurities of 5BV, flow velocity is 1.8BV/h, finally uses 50% ethanol solution of 5BV
Wash-out, (now pH is about 3), flow velocity is 1.8BV/h.
In the present invention, double-aqueous phase system used consists of concentration of alcohol is 35-45% (w/v), and ammonium sulfate concentrations are
15-25% (w/v), remaining is water.
Preferably, in double-aqueous phase system, concentration of alcohol is 39% (w/v), and ammonium sulfate concentrations are 21% (w/v), and remaining is
Water.
Wherein, in chromatographic column, the methanol solution using 1.5～2BV is eluted.
Brief description
Fig. 1 is meoh eluate thin-layer chromatography result figure；
Fig. 2 is cordycepin standard items high performance liquid chromatography detection figure；
Fig. 3 is the cordycepin liquid high performance liquid chromatography detection figure isolated；
Fig. 4 is cordycepin crystal electron microscope.
Specific embodiment
Embodiment 1：Prepare cordycepin crystal as steps described below：
(1) Cordyceps militaris stroma is cultivated remaining matrix natural drying, pulverized and then 80 mesh sieves excessively are standby；
(2) weigh 20g Cordyceps militaris powder, add the ethanol solution of 5 times of volumes 50%, alcohol bath extraction under the conditions of 60 DEG C
5h；
(3) it is centrifuged 10min under the conditions of 3000r/min with supercentrifuge, collect supernatant standby；
(4) with macroreticular resin, the supernatant of collected after centrifugation is purified, sample liquid and NKAII macroreticular resin are with 50ml
: the amount loading of 1g, pH 5,1.8BV/h, then use the distilled water removal of impurities of 5BV, flow velocity is 1.8BV/h, finally use the 50% of 5BV
Ethanol solution elutes, pH 3, and flow velocity is 1.8BV/h；
(5) ethanol eluate and ammonium sulfate is used to form double-aqueous phase system, wherein concentration of alcohol is 39% (w/v), ammonium sulfate
Concentration is 21% (w/v), collects supernatant and is repeatedly enriched with；
(6) launch upper on lamellae, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatographic column, with the methanol solution wash-out of 2BV, collect eluent；
(8) eluent vacuum distillation is concentrated 1/5 for original volume；
(9) the eluent 2BV methanol-eluted fractions after concentrating, collect eluent, are crystallized at room temperature and recrystallized system
Obtain cordycepin crystal 0.79g, gained crystal purity reaches 98.9%.
Embodiment 2：Prepare cordycepin crystal as steps described below：
(1) Cordyceps militaris stroma is cultivated remaining matrix natural drying, pulverized and then 80 mesh sieves excessively are standby；
(2) weigh 20g Cordyceps militaris powder, add the ethanol solution of 6 times of volumes 60%, alcohol bath extraction under the conditions of 60 DEG C
4h；
(3) it is centrifuged 15min under the conditions of 3000r/min with supercentrifuge, collect supernatant standby；
(4) with macroreticular resin, the supernatant of collected after centrifugation is purified, sample liquid and NKAII macroreticular resin are with 50ml
: the amount loading of 1.2g, 1.8BV/h, then use the distilled water removal of impurities of 5BV, flow velocity is 1.8BV/h, finally uses 50% ethanol of 5BV
Eluant solution, flow velocity is 1.8BV/h；
(5) ethanol eluate and ammonium sulfate is used to form double-aqueous phase system, wherein concentration of alcohol is 42% (w/v), ammonium sulfate
Concentration is 25% (w/v), collects supernatant and is repeatedly enriched with；
(6) launch upper on lamellae, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatographic column, with the methanol solution wash-out of 2BV, collect eluent；
(8) eluent vacuum distillation is concentrated 1/5 for original volume；
(9) the eluent 1.5BV methanol-eluted fractions after concentrating, collect eluent, are crystallized at room temperature and recrystallize
Prepared cordycepin crystal 0.82g, gained crystal purity reaches 99.8%.
Embodiment 3：Prepare cordycepin crystal as steps described below：
(1) Cordyceps militaris stroma is cultivated remaining matrix natural drying, pulverized and then 80 mesh sieves excessively are standby；
(2) weigh 20g Cordyceps militaris powder, add the ethanol solution of 4 times of volumes 70%, alcohol bath extraction under the conditions of 60 DEG C
6h；
(3) it is centrifuged 15min under the conditions of 3000r/min with supercentrifuge, collect supernatant standby；
(4) with macroreticular resin, the supernatant of collected after centrifugation is purified, sample liquid and NKAII macroreticular resin are with 50ml
: the amount loading of 0.8g, 1.5BV/h, then use the distilled water removal of impurities of 4BV, flow velocity is 1.6BV/h, finally uses 40% ethanol of 7BV
Eluant solution, flow velocity is 2BV/h；
(5) ethanol eluate and ammonium sulfate is used to form double-aqueous phase system, wherein concentration of alcohol is 37% (w/v), ammonium sulfate
Concentration is 20% (w/v), collects supernatant and is repeatedly enriched with；
(6) launch upper on lamellae, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatographic column, with the methanol solution wash-out of 1.5BV, collect eluent；
(8) eluent vacuum distillation is concentrated 1/5 for original volume；
(9) the eluent 2BV methanol-eluted fractions after concentrating, collect eluent, are crystallized at room temperature and recrystallized system
Obtain cordycepin crystal 0.78g, gained crystal purity reaches 98.3%.
In the above-mentioned methods, last crystallization and recrystallizing technology adopt common extract crystallization processes, for example permissible
Using following techniques：Eluent is crystallized at 0～4 DEG C, with vacuum filtration method, crystal is isolated, after being dried, obtain cordycepin
Crystal crude product；The ethanol solution that cordycepin crystal crude product is dissolved in 75-85% (V/V) is recrystallized, and then vacuum filtration divides
Cordycepin crystal is obtained after recrystallized product, washing and drying.
The detection method of above-mentioned products therefrom following () taking the crystal of embodiment 1 as a example：
(1) thin layer detection：
Sample drop is added on lamellae, solvent be (chloroform: isopropanol: ethyl acetate: water=8: 6: 2: 0.3),
Developed the color under 254nm ultraviolet irradiation, meoh eluate thin-layer chromatography result is shown in Fig. 1.
(2) high effective liquid chromatography for measuring cordycepin content：
Fixing phase is C18 pillar；Methyl alcohol in mobile phase: water=20: 80 (v/v)；Flow rate, 1.0ml/min；Applied sample amount
For 10 μ l, sample is using the membrane filtration front using 0.45 μm.Finally carry out ultraviolet detection under the conditions of 260nm.The efficient liquid of standard items
Phase chromatogram detection figure is shown in Fig. 2, and sample high performance liquid chromatography detection figure is shown in Fig. 3.
(3) by the cordycepin crystal obtaining in electric Microscopic observation, result is shown in Fig. 4.
Claims (7)
1. the preparation method of cordycepin crystal is it is characterised in that comprise the steps：1) to cultivate the remaining base of Cordyceps militaris stroma
Matter is raw material, is extracted with organic solvent, supernatant is collected by centrifugation after matrix is pulverized；2) utilize macroporous resin adsorption chromatogram
Post purifies to the thick liquid after centrifugation；3) step 2) gained cordycepin using ethanol, ammonium sulfate composition double-aqueous phase system enter
Row is enriched with and collects sample, and in double-aqueous phase system, concentration of alcohol is 35-45% (w/v), and ammonium sulfate concentrations are 15-25% (w/
V), remaining is water；4) fill chromatographic column, methanol solution elutes, and eluent vacuum distillation concentrates, the eluant methanol wash-out of concentration,
Eluent crystallization obtains cordycepin crystal.
2. preparation method according to claim 1 it is characterised in that the remaining matrix of described culture Cordyceps militaris stroma refer to for
The solid medium of culture Cordyceps militaris stroma removes the remaining matrix of Cordyceps militaris stroma after the completion of culture.
3. preparation method according to claim 1 crosses 80 mesh sieves it is characterised in that described matrix pulverizes standard for after pulverizing.
4. preparation method according to claim 1 it is characterised in that organic solvent used be ethanol solution, the volume weight with matrix
Amount ratio is for 2-10: 1, extraction temperature is 50-80 DEG C, and extraction time is 3-8 hour.
5. preparation method according to claim 1 is it is characterised in that chromatography parameter is cordycepin sample liquid and macropore tree
Fat is with 50ml：The amount loading of 0.8-1.5g, flow velocity is 1.5-2.0BV/h, then uses the distilled water removal of impurities of 3-8BV, and flow velocity is
1.5-2.0BV/h, finally uses the ethanol solution wash-out of 3-8BV, and flow velocity is 1.5-2.0BV/h.
6. preparation method according to claim 1 is it is characterised in that in double-aqueous phase system, concentration of alcohol is 39% (w/v), sulfuric acid
Ammonium concentration is 21% (w/v), and remaining is water.
7. preparation method according to claim 1 it is characterised in that in chromatographic column the methanol solution using 1.5～2BV washed
De-.
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| CN105111264A (en) * | 2015-08-16 | 2015-12-02 | 深圳市倍昂生物科技有限公司 | 3'-deoxyadenosine crystal form and preparation method therefor |
| CN110432258B (en) * | 2019-07-17 | 2021-09-28 | 西北农林科技大学 | Application of cordycepin in preparation of diluent for cryopreservation of goat sperm and preparation method of cordycepin |
| CN110693898A (en) * | 2019-11-23 | 2020-01-17 | 吉林省蔚来生物科技有限公司 | A pharmaceutical composition containing ginsenoside |
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| CN101157712B (en) * | 2007-11-16 | 2011-01-19 | 上海市农业科学院 | Method for separating and purifying cordycepin |
| CN101928316A (en) * | 2010-07-23 | 2010-12-29 | 上海国宝企业发展中心 | Preparation method of high-purity plate-like cordycepin crystal |
| KR20120077691A (en) * | 2010-12-31 | 2012-07-10 | 안성구 | A cordycepin producing method using unhatched egg |
| JP5850497B2 (en) * | 2011-11-30 | 2016-02-03 | 国立大学法人福井大学 | Method for producing and purifying cordycepin |
| CN103772460B (en) * | 2014-02-14 | 2016-02-10 | 江苏省中国科学院植物研究所 | The extraction process of cordycepin in silkworm Chinese caterpillar fungus |
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Granted publication date: 20170222 Termination date: 20180928 |